PDBsum entry 2px0

Biosynthetic protein

PDB id: 2px0

Contents

Protein chains

(+ 2 more) 258 a.a.

Ligands

GNP ×8

Metals

_MG ×8

Waters ×24

References listed in PDB file

Key reference

Title

The crystal structure of the third signal-Recognition particle gtpase flhf reveals a homodimer with bound gtp.

Authors

G.Bange, G.Petzold, K.Wild, R.O.Parlitz, I.Sinning.

Ref.

Proc Natl Acad Sci U S A, 2007, 104, 13621-13625. [DOI no: 10.1073/pnas.0702570104]

PubMed id

17699634

Abstract

Flagella are well characterized as the organelles of locomotion and allow bacteria to react to environmental changes. The assembly of flagella is a multistep process and relies on a complex type III export machinery located in the cytoplasmic membrane. The FlhF protein is essential for the placement and assembly of polar flagella and has been classified as a signal-recognition particle (SRP)-type GTPase. SRP GTPases appeared early in evolution and form a unique subfamily within the guanine nucleotide binding proteins with only three members: the signal sequence-binding protein SRP54, the SRP receptor FtsY, and FlhF. We report the crystal structures of FlhF from Bacillus subtilis in complex with GTP and GMPPNP. FlhF shares SRP GTPase-specific features such as the presence of an N-terminal alpha-helical domain and the I-box insertion. It forms a symmetric homodimer sequestering a composite active site that contains two head-to-tail arranged nucleotides similar to the heterodimeric SRP-targeting complex. However, significant differences to the GTPases of SRP and the SRP receptor include the formation of a stable homodimer with GTP as well as severe modifications and even the absence of motifs involved in regulation of the other two SRP GTPases. Our results provide insights into SRP GTPases and their roles in two fundamentally different protein-targeting routes that both rely on efficient protein delivery to a secretion channel.

Figure 1.
Fig. 1. Structure of FlhF in comparison with the SRP-targeting complex. (a) Domain structure of the SRP GTPases FlhF, SRP54, and FtsY. The positions of SRP GTPase-specific motifs, the I-box, and the conserved nucleotide-binding elements (G1–G5) are indicated. (b) Ribbon representation of the FlhF homodimer (green, Left) with two GTP molecules viewed perpendicular to the 2-fold axis (dashed line) and the SRP/SR heterodimer from T. aquaticus (blue, Right) with two GMPPCP molecules (21). The N domains of the two complexes are on top and the G domains at the bottom. Three motifs involved in domain communication in the SRP-targeting complex are in yellow. In FlhF these motifs are absent. In the FlhF homodimer, the N domains are not part of the dimer interface (monomers are labeled FlhF and FlhF') and are separated by 12 Å. The G domains of FlhF and FlhF' form a composite active site harboring two nucleotides similar to the SRP/SR heterodimer. (c) Sequence alignment of regulatory motifs in SRP GTPases: "ALLEADV," "DARGG," and "GQ." FlhF from B. subtilis is compared with SRP54 (Ffh) and FtsY from S. solfataricus (Sol), T. aquaticus (Taq) and E. coli (Ec). (d) Close-up of the N/G interdomain region in the FlhF homodimer shows the position of the tyrosine insertion. In Figs. 1 Go-–3, SI Figs. 4 and 6, and SI Table 3, the T. aquaticus structure (21) is used as example for the SRP/SR heterodimer. The S. solfataricus structure (G.B. and I.S., unpublished data) gives very similar results.

Figure 3.
Fig. 3. Asymmetric conformation of the putative catalytic arginine residue in the G2 element. (a) In the structure of the FlhF/GMPPNP, both Arg-216 residues of the homodimer are well defined in an unbiased F[o] – F[c] difference density map (2.0 ; calculated without the arginines; green grid). Their asymmetric arrangement is as observed for the corresponding arginines in b for the SRP/SR heterodimer with bound GMPPCP from T. aquaticus (21). (c) Sequence alignment of G2 elements in SRP GTPases. The sequence is annotated as in Fig. 1c. Note: The crystal of the FlhF/GMPPNP complex contains eight monomers in the AU that form four noncrystallographic dimers. The asymmetry of Arg-216 is observed in all of them.